1. Field of the Invention
The present invention is directed to the identification and characterization of classes and subclasses of circulating cancer cells, including microtumors, from body fluid samples.
2. Related Art
Metastases are established potentially by cancer cells, derived from a primary or a secondary tumor site, that are released into the circulation via the blood and/or the lymphatic system. Metastatic dissemination can be an early event in the natural history of many malignant epithelial tumors. Blood serves as a passage between the primary and metastatic site that is ultimately seeded by tumor cells that survive detachment and circulation in the peripheral blood and bone marrow. The initial concept for the development of a metastasis and infiltration of blood vessels by cancer cells from a primary malignant tumor was formulated by Cruveilhier in 1829 and confirmed by Ashworth in 1869 (Ashworth, et al, Aust. Med J. 14:146 (1869)). Ashworth et al. described cells in the blood resembling those found in malignant skin tumors at autopsy, but the malignant nature of these cells was not determined with certainty.
In the blood circulation, the metastatic pathway can be developed via intravascular or extravascular growth. During the extravascular metatstatic process, the tumor cells undergo a series of sequentially-linked steps. For example, the tumor cells intravasate, survive in the circulation, embolize, arrest in distant capillaries, extravasate, and multiply in organ parenchyma to generate a secondary or tertiary tumor (Fidler, I .J., Cancer Research 50:6130-6138 (1990)).
The definitive classification of a malignant cell found in a body fluid sample remains challenging. Many early cancer researchers were cytologists whose identification criteria for cancer cells were established by the Papanocolau method id and Romanowsky stains. Such criteria for classifying a cell as malignant included the following morphological examination: abnormal cell size and shape, a large nucleus with an abnormal chromatin network, prominent (often multiple) nucleoli, scanty cytoplasm, and cytoplasmic vacuolation. Due to a previous lack of specific identification criteria for circulating cancer cells, these cells could not be easily distinguished from blood cell precursors, a typical forms of cells normally found in the blood, or cells foreign to the blood. Multiple staining methods (including fluorescent staining techniques, but without molecular markers) were advocated for the identification of circulating cancer cells (Herbeuval et al, Acta. Cytol. 305:73-82 (1965); Nagy et al., Acta. Cytol. 305:61-67 (1965)). The advent of monoclonal antibodies, nucleic acid probes, and multiple fluorescent dyes, and the development of multiple filter functions for fluorescence microscopy have aided in the elucidation of circulating cancer cells isolated from patients.
Invasive potential has been linked to chromosome aneuploidy, hormone receptors, cell proliferation markers, and proliferative cell nuclear antigen (PCNA). Chromosome aneuploidy in cancer patients and the relationship to invasiveness in clinical applications have been correlated by Wingren, et al., Br. J. Cancer 69: 546-549 (1994). Further, using flow cytometry on cells derived from breast cancer patients, Lee, et al, Mod. Pathol. 5: 61-67 (1992), found that aneuploidy was significantly related to the loss of estrogen receptors, high histologic grade, high nuclear grade and mitotic rate. Immunohistochemical evaluation of proliferation by staining with Ki67 monoclonal antibody correlated strongly with mitotic rate. Aneuploid and polyploid tumors demonstrated higher Ki67 scores than diploid tumors. Correlation was demonstrated between  aneuploidy and low levels of estrogen receptors (Fernandes, et al., Can. J. Surg. 34: 349-355 (1991)).
Invasive potential has been linked with cell proliferation markers, such as MiB1/Ki67 and proliferating cell nuclear antigen (CNA). MiB1/Ki67, introduced by Gerdes, Int. J. Cancer 31: 13-20(1983), provides a direct means of evaluating the growth fraction of tumors in histopathology and cytopathology (Key, et al, Lab. Invest. 68: 629-636 (1993)). Sasano, et al, Anticancer Res. 17:3685-3690 (1997)) found a significant correlation between the cell proliferation MiB1/Ki67 marker and invasive ductal breast carcinoma. Vielh, Am. J. Clin. Pathol 94: 681-686 (1990), conducted a study of immunohistologic staining (Ki67 index) versus flow cytometry using a Ki67 monoclonal antibody. Immunohistochemical studies provided better proliferative indices than flow cytometry. PCNA is also a good marker of cell proliferation with evidence of deregulated expression in some neoplasms and occasional upregulation in benign tissue (EL-Habashi, et al., Acta Cytol. 41:636-648 (1997); Hall, et al., J Pathol. 162:285-294 (1990); Leong and Milios, Appl. Immunohistochem 1:127-135 (1993); Siitonen, et al., Am. J. Pathol. 142:1081-1088 (1993); Galand and Degraef, Cell Tissue Kinet. 22:383-392 (1989)). Kirkegaard, et al., Astrocytomas. Anat. Pathol. 109:69-74 (1997), found in astrocytomas that proliferation, measured as MiB1/Ki67 and PCNA by image cytometry, correlated significantly with histologic grade and patient survival.
The P27/Kip proteins play an important role as negative regulators of cell cycle-dependent kinase activity during progression of the cell cycle. Tsihlias, et al., Cancer Res. 58:542-548 (1998)) found prostate cancers that had increased P27 staining were correlated with benign prostatic epithelial components in all tumor sections.
Correlation of proliferation markers, estrogen receptors, and drug therapy in circulating cells has been done with breast cancer biopsy material by Makris, et al., Breast Cancer Res. Treat. 48:11-20 (1998) in a “first-time” study where an early decrease in proliferation marker was shown to relate to the subsequent clinical response to tamoxifen therapy.
A sensitive test has been developed for enrichment of circulating cancer cells by using double gradient centrifugation and immunomagnetic cell sorting to deplete most erythrocytes and leukocytes (U.S. Pat. No. 5;962,237). Isolated circulating cancer cells are characterized using multiple identification markers, such as epithelial, tissue or cell-specific markers, chromosome aneuploidy markers, and nuclear markers (Ts'o, P. O. P., et al. Urology 49(6):881-885 (1997)).
Still, with current techniques and knowledge, the pathway of secondary tumor formation via intravascular growth of tumor cells in the circulation lacks sufficient distinction. Reasons for this general lack of understanding include the complex nature of cancer, the many different kinds of cancer, the lack of suitable technology for detecting and characterizing multistaged circulating tumor cells, and the difficulty of describing the fate of the circulating cancer cells in an intact physiological state. Molecular, cytological, and/or morphological characterization of circulating cancer cells isolated from body fluids, including peripheral blood, is designed to address these concerns and is the subject of this invention.